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Biotransformation studies on tobacco cembranoids using plant cell cultures

University of British Columbia

This thesis deals with the biotransformation studies on tobacco cembranoids using the plant cell culture lines coded as TRP4a and T-43-T which are derived from Tripterygium wilfordii, an important Chinese herbal plant, and Nicotiana sylvestris, respectively. The studies are divided into three parts: 1) Biotransformation of the diols 1 and 2 using the TRP4a cell line. 2) Biotransformation of the diol 1 using the T-43-T cell line. 3) Biotransformation of cembranoid analogues using the T-43-T cell line. Incubation of the diol 1 with the growing cells of TRP4a and with cells resuspended in phosphate (pH 6.3) or TrisHC1 (pH 7.5) buffers for varying time periods affords five products4, 8, 92, 93 and 94. The epoxide 4 is the major product occurring in about 50% yield. The allylic alcohols 92 and 93 are assigned as a pair of diastereoisomers with different chiralities at C-10 but their absolute configurations at this centre have not been established. Similarly, when the diol 2 is incubated with the growing cells of TRP4a, the epoxide 95 is obtained as a major product in about 50% yield. Therefore, it is confirmed that the 11, 12 double bond in the diols 1or 2 is the most active site for oxidative reactions. The reaction parameters such as cell age, buffer, pH, incubation time, substrate concentration and substrate administration methods have been investigated. No significant biotransformations are achieved when the cell free extract (CFE) and the cell homogenate prepared from the TRP4a cells are involved and the substrate diol 1 is recovered in each case. However, the pellet fractions obtained during the centrifugation in the preparation of CFE, when resuspended in phosphate buffer (pH 6.6) and with addition of hydrogen peroxide, FMN and manganous chloride as cofactors, are capable of transforming the diol 1 into the epoxide 4 in about 40% yield. Biotransformation studies using the T-43-T cell line derived from Nicotiana sylvestris, a tobacco species in which the diols 1 and 2 as well as many other cembranoids are encountered, indicate that the growing cells are capable of transforming the diol 1 into the epoxide 4 and the allylic alcohols, but only in low yields when compared to the data with TRP4a. The epoxide 4, which is a major product in studies with the TRP4a cell line, is a minor component (about 20%)in the product mixture resulting from experiments with T-43-T cell line. On the other hand, theC-10 and C-12 alcohols, 93 and 8 respectively, are obtained in relatively higher yields (20-29%). However, in contrast to the data obtained with TRP4a, when such cofactors as hydrogen peroxide, FMN and manganese chloride are added, both the T-43-T cell homogenate and the pellets resuspended in phosphate buffer (pH 6.6) afford good yields of the epoxide 4 (72% and 62%, respectively) in biotransformation experiments with the diol 1. Biotransformation of cembranoid analogues using the T-43-T cell line was also investigated. The growing cells can transform the epoxide 4 into the triol 109, a product formed by hydroxylation at the unactivated methine position C-15, in 55% yield. In experiments with cell homogenates obtained from the T-43-T cell line, epoxidation is observed in the enone 43 and the seco-diketone 44 and epoxides 7 and 107 are obtained respectively. On the other hand, the aldehyde 32 is reduced selectively to the alcohol 110,which in turn, undergoes a cyclization to the ether 111 in a SN2'-like manner in the presence of such cofactors as hydrogen peroxide, manganous chloride and FMN. The tetrol 104, when exposed to cell homogenate, is oxidized at C-6 and the latter intermediate undergoes a spontaneous intra-molecular Michael addition to give the ether 112 in approximately 20% yield. In addition, the ethers 113 and 114 are obtained in very small amounts. In conclusion, the T-43-T cell line is capable of performing both oxidation and reduction reactions, namely selective epoxidation of the 11, 12 double bond, hydroxylation at the allylic positions (C-10 and C-12), hydroxylation at a non-activated methine position (C-15) and selective reduction of aldehyde to the corresponding alcohol. . . . [molecular structure symbols]

Thesis/Dissertation

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2008-09-17

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http://hdl.handle.net/2429/2182

Chemistry

University of British Columbia

UBCV

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